The identification of stem cells and differentiation programs regulating the development and maintenance of the normal prostate epithelium is essential for the identification of the cell type(s) and molecular changes involved in the development and propagation of prostate cancer. The p63 gene is selectively expressed in basal cells of various epithelia and is required for epithelial development. p63 has been recently shown to be essential for the proliferative potential of stem cells both in the thymus epithelium and the epidermis. Previous work from our group and others suggests that during normal prostate development, secretory cells derive from p63-positive basal cells, which thus represent progenitor/stem cells. However, the mechanisms underlying the in vivo maintenance of the prostate epithelium during post-natal life remain to be clarified. The suggestion that basal and secretory prostate cells are hierarchically related during development raises the possibility that the renewal of the adult prostate depends on p63-positive basal stem cells. While data from both in vivo and in vitro studies provide support for such a possibility, a contrasting model proposes that adult secretory cells are capable of sustaining their own renewal, without significant contribution from basal stem cells. The experiments proposed in this application are aimed at clarifying the renewal process of the adult prostate epithelium both in physiologic conditions and after cell injury. We will first employ a novel DNA-analog-based lineage tracing method to test whether the adult prostate epithelium contains specialized stem/progenitor cells that repeatedly divide in vivo to give rise to post-mitotic cells (Aim 1). In a parallel and complementary effort, we will perform genetic lineage tracing to determine whether the in vivo development and renewal of the prostate epithelium depends on p63-positive progenitor/stem cells (Aim 2). Finally, we will begin to study the molecular mechanisms of prostate stem cell regulation by testing the hypothesis that p63 plays a central role in modulating prostate stem cell functions (Aim 3). Our proposal will provide extremely valuable knowledge on the way the normal prostate epithelium develops and is renewed in vivo. Such knowledge is absolutely required for the future identification of the cell type(s) and molecular abnormalities involved in prostate cancer development and progression.